🥜 Food Allergens: Gluten (Gliadin) — R5 ELISA

Food Chemistry AOAC 2012.01

Overview

STATUS (as of 06.11.2026). AOAC Official Method 2012.01 — determination of gliadin as a measure of gluten by the R5 sandwich ELISA (kit with the R5 antibody and “cocktail” extraction, the R5 Méndez method). The method was adopted as First Action in 2012 with an “in foods” claim; at Final Action status (2016) the claim was reduced to rice- and corn-based matrices, and after a collaborative study with 14 laboratories (J. AOAC Int. 2022, vol. 105, issue 2) the title and applicability statement were changed, extending it among others to soy, starches, pseudocereals, legumes, spices, juices, meat, fish, bread, cookies and potatoes. The same R5 method is a Codex Alimentarius type I method (CXS 118-1979). AOAC 2012.01 concerns only gluten (prolamins of wheat, rye and barley) — it does not cover other allergens. Other AOAC designations listed so far next to it are separate methods: AOAC 991.19 is the Skerritt ELISA, and number 2014.03 is held by a kit with the G12 antibody.

PRINCIPLE. The wells of the microplate are coated with the R5 antibody against gliadins. Gliadin from the standard solution or the sample binds to the antibody; after washing, R5 antibody conjugated to peroxidase is added and binds to the complex (“sandwich” arrangement). After another wash, substrate and chromogen are added — the bound conjugate converts the colourless chromogen into a blue product, and the stop solution changes the colour to yellow. The absorbance is measured at 450 nm; it is proportional to the gliadin concentration (ng/ml).

SAMPLE PREPARATION. The sample is homogenised in an amount ensuring a representative portion (e.g. 50 g). A test portion of 0.25 g (liquids 0.25 ml) is mixed with 2.5 ml of “cocktail” and extracted for 40 min in a water bath at 50 °C, after cooling 7.5 ml of 80 % ethanol is added and shaken for 1 h at room temperature, then filtered or centrifuged for 10 min at at least 2500 × g. Food with tannins and polyphenols (e.g. chocolate, coffee, cocoa, fruit juices, spices, buckwheat) is extracted with the addition of 0.25 g of skimmed milk powder; oats — from a 1 g test portion with four times the amount of reagents. The extract is diluted with buffer 1:12.5, giving a total dilution factor of 500.

CALCULATION. The gliadin concentration read from the standard curve (standards 0, 5, 10, 20, 40 and 80 ng/ml) is multiplied by the dilution factor 500, and the gliadin result — by 2, because according to the Codex Alimentarius prolamins make up 50 % of gluten. Example from the instructions: 10 ng/ml → 5 mg/kg gliadin → 10 mg/kg gluten. Results between the limit of detection and the limit of quantification are reported qualitatively (“< LoQ”).

REGULATIONS. Regulation (EU) No 1169/2011, Annex II — 14 groups of substances causing allergies or intolerances, including cereals containing gluten (wheat, rye, barley, oats, spelt, kamut). Implementing Regulation (EU) No 828/2014 — “gluten-free”: not more than 20 mg/kg of gluten in the food as sold to the consumer; “very low gluten”: not more than 100 mg/kg (cereal ingredients specially processed to reduce gluten); oats in such food — not more than 20 mg/kg of gluten. Codex Alimentarius CXS 118-1979: the same thresholds of 20 and 100 mg/kg, determination by an immunological method with a detection limit of 10 mg gluten/kg or below, method — R5 Méndez ELISA.

WHAT WE DO NOT GIVE HERE. The method is not suitable for quantifying fermented or hydrolysed gluten. It does not cover the determination of other allergens (milk, eggs, peanuts, soy, nuts, sesame and others) — these are determined with other kits, according to their manufacturers’ instructions; general principles of immunological methods are given in EN 15633-1. Of the precision from the collaborative study we give only the range of recoveries and reproducibility from the publication (recovery 80–130 %, relative reproducibility standard deviation 9.8–27.7 %).

Method principle

Sandwich ELISA with the monoclonal R5 antibody: gliadin from the extract binds to the antibody on the well walls and then to the R5 antibody conjugated to peroxidase. The bound conjugate converts the chromogen into a coloured product; after the reaction is stopped, the absorbance is measured at 450 nm. The gliadin concentration is read from the standard curve, multiplied by the dilution factor, and gluten is calculated as gliadin × 2.

Applications

• Verification of “gluten-free” (≤ 20 mg/kg gluten) and “very low gluten” (≤ 100 mg/kg) labelling — Implementing Regulation (EU) No 828/2014
• Food for people intolerant to gluten — Codex Alimentarius type I method (CXS 118-1979)
• Detection of gliadin and related prolamins of rye and barley in food declared gluten-free: rice- and corn-based products, soy, starches, pseudocereals, legumes, spices, juices, meat, fish, bread, cookies, confectionery, potatoes (scope of AOAC 2012.01 after the 2022 extension)
• Allergen information — cereals containing gluten as one of the 14 groups of Annex II to Regulation (EU) No 1169/2011

Key parameters

Standard

Standard number

AOAC Official Method 2012.01

Title (PL)

Gliadyna jako miara glutenu w żywności — kanapkowa ELISA z przeciwciałem R5 (tłumaczenie robocze)

Title (EN)

Gliadin as a measure of gluten in food — R5 sandwich ELISA

Step-by-step procedure

1. Prevention of contamination

   Work with gloves; surfaces and equipment cleaned with 40 % ethanol or 2-propanol; sample preparation in a room separated from the ELISA work.

2. Homogenisation

   Grinding and mixing of an amount ensuring a representative portion (e.g. 50 g); oats — at least 200 g.

3. “Cocktail” extraction

   0.25 g (liquids 0.25 ml) + 2.5 ml of “cocktail”, mixing, 40 min in a water bath at 50 °C; for tannins and polyphenols addition of 0.25 g of skimmed milk powder; oats — 1 g and 10 ml.

   ⏱ Time: 40 min • 🌡 Temperature: 50 °C

4. Ethanol and shaking

   Brief cooling (1–3 min), 7.5 ml of 80 % ethanol (oats 30 ml), shaking for 1 h at room temperature.

   ⏱ Time: 1 h • 🌡 Temperature: 20–25 °C

5. Clarification

   Filtration or centrifugation for 10 min at at least 2500 × g; supernatant into a clean vessel. The undiluted extract can be stored in the dark at room temperature for up to 8 weeks.

6. Dilution

   Extract 1:12.5 with buffer (e.g. 80 µl + 920 µl) — total dilution factor 500; use within 30 min.

7. Incubation with sample

   100 µl of standards and samples in duplicate, 30 min at room temperature; washing three times with 250 µl of wash buffer.

   ⏱ Time: 30 min

8. Incubation with conjugate

   100 µl of diluted conjugate, 30 min at room temperature; washing as above.

   ⏱ Time: 30 min

9. Colour reaction

   50 µl of substrate and 50 µl of chromogen, 30 min at room temperature in the dark; then 100 µl of stop solution.

   ⏱ Time: 30 min

10. Measurement and calculation

    Absorbance at 450 nm within 30 min; concentration from the standard curve × 500 = mg/kg gliadin; × 2 = mg/kg gluten. Results above standard 6 — new dilution of the extract.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• The test is carried out only by trained laboratory personnel, strictly following the instructions
• The extraction “cocktail” is harmful to health — it contains mercaptoethanol; work in a fume hood and avoid skin contact (gloves)
• The kit may contain hazardous substances — hazards according to the manufacturer’s safety data sheets
• Reagents and materials after use — recovered or disposed of according to national waste regulations

Frequently asked questions